Translationally controlled tumor protein interacts with TaCIPK23 to positively regulate wheat resistance to Puccinia triticina.

Hypersensitive response-programmed cell death (HR-PCD) regulated by Ca2+ signal is considered the major regulator of resistance against Puccinia triticina (Pt.) infection in wheat. In this study, the bread wheat variety Thatcher and its near-isogenic line with the leaf rust resistance locus Lr26 were infected with the Pt. race 260 to obtain the compatible and incompatible combinations, respectively. The expression of translationally controlled tumor protein (TaTCTP) was upregulated upon infection with Pt., through a Ca2+-dependent mechanism in the incompatible combination. The knockdown of TaTCTP markedly increased the area of dying cell and the number of Pt. haustorial mother cells (HMCs) at the infection sites, whereas plants overexpressing the gene exhibited enhanced resistance. The interaction between TaTCTP and calcineurin B-like protein-interacting protein kinase 23 (TaCIPK23) was also investigated, and the interaction was found occurred in the endoplasmic reticulum. TaCIPK23 phosphorylated TaTCTP in vitro. The expression of a phospho-mimic TaTCTP mutant in Nicotiana benthamiana promoted HR-like cell death. Silencing TaCIPK23 or TaCIPK23/TaTCTP co-silencing resulted in the same results as silencing TaTCTP. This suggested that TaTCTP is a novel phosphorylation target of TaCIPK23, and both participate in the resistance of wheat to Pt. in the same pathway.

TaCAMTA4 negatively regulates H2O2-dependent wheat leaf rust resistance by activating catalase 1 expression.

Leaf rust, caused by Puccinia triticina Erikss. (Pt), is a serious disease threatening wheat (Triticum aestivum L.) production worldwide. Hydrogen peroxide (H2O2) triggered by Pt infection in resistant wheat cultivars cause oxidative damage directly to biomolecules or is activated by calcium signaling and mediates the hypersensitive response. Calmodulin-binding transcriptional activator 4 (TaCAMTA4) has been reported to negatively regulate wheat resistance to Pt. In this study, we found that TaCAMTA4 was induced by Pt race 165 in its compatible host harboring the Pt resistant locus Lr26, TcLr26, and silencing of TaCAMTA4 increased local H2O2 accumulation and Pt resistance. Subcellular localization and autoactivation tests revealed that TaCAMTA4 is a nucleus-localized transcriptional activator. Furthermore, four DNA motifs recognized by TaCAMTA4 were identified by transcription factor-centered Y1H. Through analyzing the transcriptome database, four gene clusters were identified, each containing a different DNA motif on each promoter. Among them, the expression of catalase 1 (TaCAT1) with motif-1 was highly induced in the compatible interaction and was decreased when TaCAMTA4 was silenced. The results of EMSA, ChIP-qPCR, and RT-qPCR further showed that TaCAMTA4 directly bound motif-1 in the TaCAT1 promoter. Furthermore, silencing of TaCAT1 resulted in enhanced resistance to Pt and increased local H2O2 accumulation in wheat, which is consistent with that of TaCAMTA4. Since CAMTAs are Ca2+ sensors and catalases catalyze the decomposition of H2O2, we hypothesize that Ca2+ regulates the plant immune networks that are controlled by H2O2 and implicate a potential mechanism for Pt to suppress resistance by inducing the expression of the TaCAMTA4-TaCAT1 module, which consequently enhances H2O2 scavenging and attenuates H2O2-dependent resistance.

A comprehensive analysis of the WRKY family in soybean and functional analysis of GmWRKY164-GmGSL7c in resistance to soybean mosaic virus.

BACKGROUND: Soybean mosaic disease caused by soybean mosaic virus (SMV) is one of the most devastating and widespread diseases in soybean producing areas worldwide. The WRKY transcription factors (TFs) are widely involved in plant development and stress responses. However, the roles of the GmWRKY TFs in resistance to SMV are largely unclear. RESULTS: Here, 185 GmWRKYs were characterized in soybean (Glycine max), among which 60 GmWRKY genes were differentially expressed during SMV infection according to the transcriptome data. The transcriptome data and RT-qPCR results showed that the expression of GmWRKY164 decreased after imidazole treatment and had higher expression levels in the incompatible combination between soybean cultivar variety Jidou 7 and SMV strain N3. Remarkably, the silencing of GmWRKY164 reduced callose deposition and enhanced virus spread during SMV infection. In addition, the transcript levels of the GmGSL7c were dramatically lower upon the silencing of GmWRKY164. Furthermore, EMSA and ChIP-qPCR revealed that GmWRKY164 can directly bind to the promoter of GmGSL7c, which contains the W-box element. CONCLUSION: Our findings suggest that GmWRKY164 plays a positive role in resistance to SMV infection by regulating the expression of GmGSL7c, resulting in the deposition of callose and the inhibition of viral movement, which provides guidance for future studies in understanding virus-resistance mechanisms in soybean.

A Glycine max sodium/hydrogen exchanger enhances salt tolerance through maintaining higher Na+ efflux rate and K+/Na+ ratio in Arabidopsis.

BACKGROUND: Soybean (Glycine max (L.)) is one the most important oil-yielding cash crops. However, the soybean production has been seriously restricted by salinization. It is therefore crucial to identify salt tolerance-related genes and reveal molecular mechanisms underlying salt tolerance in soybean crops. A better understanding of how plants resist salt stress provides insights in improving existing soybean varieties as well as cultivating novel salt tolerant varieties. In this study, the biological function of GmNHX1, a NHX-like gene, and the molecular basis underlying GmNHX1-mediated salt stress resistance have been revealed. RESULTS: We found that the transcription level of GmNHX1 was up-regulated under salt stress condition in soybean, reaching its peak at 24 h after salt treatment. By employing the virus-induced gene silencing technique (VIGS), we also found that soybean plants became more susceptible to salt stress after silencing GmNHX1 than wild-type and more silenced plants wilted than wild-type under salt treatment. Furthermore, Arabidopsis thaliana expressing GmNHX1 grew taller and generated more rosette leaves under salt stress condition compared to wild-type. Exogenous expression of GmNHX1 resulted in an increase of Na+ transportation to leaves along with a reduction of Na+ absorption in roots, and the consequent maintenance of a high K+/Na+ ratio under salt stress condition. GmNHX1-GFP-transformed onion bulb endothelium cells showed fluorescent pattern in which GFP fluorescence signals enriched in vacuolar membranes. Using the non-invasive micro-test technique (NMT), we found that the Na+ efflux rate of both wild-type and transformed plants after salt treatment were significantly higher than that of before salt treatment. Additionally, the Na+ efflux rate of transformed plants after salt treatment were significantly higher than that of wild-type. Meanwhile, the transcription levels of three osmotic stress-related genes, SKOR, SOS1 and AKT1 were all up-regulated in GmNHX1-expressing plants under salt stress condition. CONCLUSION: Vacuolar membrane-localized GmNHX1 enhances plant salt tolerance through maintaining a high K+/Na+ ratio along with inducing the expression of SKOR, SOS1 and AKT1. Our findings provide molecular insights on the roles of GmNHX1 and similar sodium/hydrogen exchangers in regulating salt tolerance.

Reliable assessment and prediction of moderate preoxidation of sodium hypochlorite for algae-laden water treatment.

Chemical moderate preoxidation for algae-laden water is an economical and prospective strategy for controlling algae and exogenous pollutants, whereas it is constrained by a lack of effective on-line evaluation and quick-response feedback method. Herein, excitation-emission matrix parallel factor analysis (EEM-PARAFAC) was used to identify cyanobacteria fluorophores after preoxidation of sodium hypochlorite (NaClO) at Excitation/Emission wavelength of 260(360)/450 nm, based on which the algal cell integrity and intracellular organic matter (IOM) release were quantitatively assessed. Machine learning modeling of fluorescence spectral data for prediction of moderate preoxidation using NaClO was established. The optimal NaClO dosage for moderate preoxidation depended on algal density, growth phases, and organic matter concentrations in source water matrices. Low doses of NaClO (<0.5 mg/L) led to short-term desorption of surface-adsorbed organic matter (S-AOM) without compromising algal cell integrity, whereas high doses of NaClO (≥0.5 mg/L) quickly caused cell damage. The optimal NaClO dosage increased from 0.2-0.3 mg/L to 0.9-1.2 mg/L, corresponding to the source water with algal densities from 0.1 × 106 to 2.0 × 106 cells/mL. Different growth stages required varying NaClO doses: stationary phase cells needed 0.3-0.5 mg/L, log phase cells 0.6-0.8 mg/L, and decaying cells 2.0-2.5 mg/L. The presence of natural organic matter and S-AOM increased the NaClO dosage limit with higher dissolved organic carbon (DOC) concentrations (1.00 mg/L DOC required 0.8-1.0 mg/L NaClO, while 2.20 mg/L DOC required 1.5-2.0 mg/L). Compared to other predictive models, the machine learning model (Gaussian process regression-Matern (0.5)) performed best, achieving R2 values of 1.000 and 0.976 in training and testing sets. Optimal preoxidation followed by coagulation effectively removed algal contaminants, achieving 91%, 92%, and 92% removal for algal cells, turbidity, and chlorophyll-a, respectively, thereby demonstrating the effectiveness of moderate preoxidation. This study introduces a novel approach to dynamically adjust NaClO dosage by monitoring source water qualities and tracking post-preoxidation fluorophores, enhancing moderate preoxidation technology application in algae-laden water treatment.

Management of take-all disease caused by Gaeumannomyces graminis var. tritici in wheat through Bacillus subtilis strains.

Wheat (Triticum aestivum) is the second largest grain crop worldwide, and one of the three major grain crops produced in China. Take-all disease, caused by Gaeumannomyces graminis var. tritici (Ggt) infection, is a widespread and devastating soil-borne disease that harms wheat production. At present, the prevention and control of wheat take-all depend largely on the application of chemical pesticides. Chemical pesticides, however, not only lead to increased drug resistance of pathogens but also leave significant residues in the soil, causing serious environmental pollution. In this study, we investigated the application of Bacillus subtilis to achieve take-all disease control in wheat while reducing pesticide application. Antagonistic bacteria were screened by plate test, species identification of strains was performed by Gram staining and sequencing of 16s rDNA, secondary metabolite activity of strains was detected by clear circle method, strain compatibility and effect of compounding on Ggt were detected by plate, and the application prospects of specific strains were analyzed by greenhouse and field experiments. We found that five B. subtilis strains, JY122, JY214, ZY133, NW03, Z-14, had significant antagonistic effects against Ggt, and could secrete antimicrobial proteins including amylase, protease, and cellulase. Furthermore, Z-14 and JY214 cultures have also been shown to change the morphology of Ggt mycelium. These results also showed that Z-14, JY214, and their combination can control take-all disease in wheat at a reduced level of pesticide use. In summary, we screened two Bacillus spp. strains, Z-14 and JY214, that could act as antagonists that contribute to the biological control of wheat take-all disease. These findings provide resources and ideas for controlling crop diseases in an environmentally friendly manner.

Callose deposited at soybean sieve element inhibits long-distance transport of Soybean mosaic virus.

The function of callose and its deposition characteristics at phloem in the resistance to the long-distance transportation of Soybean mosaic virus (SMV) through phloem was studied. Two different methods of SMV inoculation were used in the study, one was direct friction of the virus on seedling leaves and the other was based on grafting scion and rootstock to create different resistance and sensitivity combinations. Veins, petioles of inoculated leaves and rootstock stems were stained with callose specific dye. Results from fluorescence microscope observation, pharmacological test, and PCR detection of SMV coat protein gene (SMV-CP) showed the role of callose in long-distance transportation of SMV through phloem during infection of soybean seedlings. When the inhibitor of callose synthesis 2-deoxy-D-glucose (2-DDG) was used, the accumulation of callose fluorescence could hardly be detected in the resistant rootstocks. These results indicate that callose deposition in phloem restricts the long-distance transport of SMV, and that the accumulation of callose in phloem is a main contributing factor for resistance to this virus in soybean.

A Golgi-Localized Sodium/Hydrogen Exchanger Positively Regulates Salt Tolerance by Maintaining Higher K+/Na+ Ratio in Soybean.

Salt stress caused by soil salinization, is one of the main factors that reduce soybean yield and quality. A large number of genes have been found to be involved in the regulation of salt tolerance. In this study, we characterized a soybean sodium/hydrogen exchanger gene GmNHX5 and revealed its functional mechanism involved in the salt tolerance process in soybean. GmNHX5 responded to salt stress at the transcription level in the salt stress-tolerant soybean plants, but not significantly changed in the salt-sensitive ones. GmNHX5 was located in the Golgi apparatus, and distributed in new leaves and vascular, and was induced by salt treatment. Overexpression of GmNHX5 improved the salt tolerance of hairy roots induced by soybean cotyledons, while the opposite was observed when GmNHX5 was knockout by CRISPR/Cas9. Soybean seedlings overexpressing GmNHX5 also showed an increased expression of GmSOS1, GmSKOR, and GmHKT1, higher K+/Na+ ratio, and higher viability when exposed to salt stress. Our findings provide an effective candidate gene for the cultivation of salt-tolerant germplasm resources and new clues for further understanding of the salt-tolerance mechanism in plants.

[Interaction between wheat translationally controlled tumor protein TCTP and SNF1-related protein kinase SnRK1].

Translationally controlled tumor proteins (TCTP) and SNF1- related protein kinase (SnRK1) are conserved and widely present in eukaryotic cells. TCTP regulates cell division, plant growth and development, and mediates plant resistance against pathogen infection. SnRK1 participates in a range of physiological processes including sugar metabolism and resistance to abiotic and biotic stresses. Previous work in our laboratory demonstrated that wheat TCTP can respond to Puccinia triticina infection and induce host defense responses. In order to further investigate the mechanism of TaTCTP in wheat resistance to Puccinia triticina infection, we used TAP (tandem affinity purification) and mass spectrometry to screen the potential interactants of TaTCTP. A SNF1- related protein kinase (SnRK1) was identified as a potential interacting protein of TaTCTP. The results of yeast two-hybrid assay showed that TCTP could interact with SnRK1 in yeast, and the yeast carrying TCTP and SnRK1 could grow on SD/-Leu/-Trp/-His/-Ade (SD/-LWHA) medium. The fluorescence signal of the interaction between TCTP and SnRK1 was found to be distributed in the cytoplasm in the Bi-fluorescense complementation experiment. Co-IP experiments further showed that TCTP and SnRK1 could interact in plant cells. This study lays an important foundation for further studying the mechanism of TaTCTP in the interaction between wheat and Puccinia triticina, and it play a great influence on further improving the molecular mechanism of wheat resistant to Puccinia triticina.

Changes of Nitric Oxide and Its Relationship with H2O2 and Ca2+ in Defense Interactions between Wheat and Puccinia Triticina.

In this research, the wheat cultivar 'Lovrin 10' and Puccinia triticina races 165 and 260 were used to constitute compatible and incompatible combinations to investigate the relationship between NO and H2O2 and between NO and calcium (Ca(2+)) signaling in the cell defense process by pharmacological means. The specific fluorescent probe DAF-FM DA was coupled with confocal laser scanning microscopy and used to label intracellular nitric oxide (NO) and monitoring the real-time NO dynamics during the processes of wheat defense response triggered by P. triticina infection. The results showed that at 4 h after inoculation, weak green fluorescence was observed in the stomatal guard cells at the P. triticina infection site in the incompatible combination, which indicates a small amount of NO production. Twelve hours after inoculation, the fluorescence of NO in- cell adjacent to the stomata gradually intensified, and the NO fluorescent area also expanded continuously; the green fluorescence primarily occurred in the cells undergoing a hypersensitive response (HR) at 24-72 h after inoculation. For the compatible combination, however, a small amount of green fluorescence was observed in stomata where the pathogenic contact occurred at 4 h after inoculation, and fluorescence was not observed thereafter. Injections of the NO scavenger c-PTIO prior to inoculation postponed the onset of NO production to 48 h after inoculation and suppressed HR advancement. The injection of imidazole, a NADPH oxidase inhibitor, or EGTA, an extracellular calcium chelator, in the leaves prior to inoculation, delayed the onset of NO production in the incompatible combination and suppressed HR advancement. Combined with our previous results, it could be concluded that, Ca(2+) and hydrogen peroxide (H2O2) are involved in upstream of NO production to induce the HR cell death during P. triticina infection, and Ca(2+), NO and H2O2 are jointly involved in the signal transduction process of HR in the interaction system.